The present invention relates generally to the synthesis of nucleic acids and more particularly relates to solid supports that are useful in automated synthesis of DNA or RNA oligonucleotides.
Universal Solid supports used in automated oligonucleotide synthesis possess pre-attached nucleosides to provide a chain initiation site for oligonucleotide construction. Chain elongation occurs by sequential addition of monomeric phosphoramidite unites on the 5xe2x80x2 hydroxyl. During the deprotection process of the oligodeoxyribonucleotide, the pre-attached nucleoside is cleaved from the solid support and retained on the oligodeoxyribonucleotide as the 3xe2x80x2-terminated base. Solid supports suitable for automated synthesis of oligonucleotides must satisfy the following characteristics:
1) the solid support must react selectively with the functionalized 3xe2x80x2 end of the nucleotide in particular of the phosphoramidite, H-phosphonate, phosphotiester, phosphodiester, phosphite type or with any other monomer reagent according to the synthetics method used;
2) the support-oligonucleotide bond must be stable under the conditions of the synthesis;
3) the support oligonucleotide bond must be able to be hydrolyzed at the end of the synthesis under the conditions for the step of deprotection of the oligonucleotide; and
4) the covalent bond between the support and oligonucleotide must be such that, during the detachment, the released oligonucleotide must be such that, during the detachment, the release oligonucleotide is of native type, that is to say that the 3xe2x80x2 terminal hydroxyl function is free or does not bear any residue derived from the synthesis.
Many supports have already been described in the literature for the solid phase synthesis of oligonucleotides. These supports may consist of organic polymers such as polystyrene (Nucleic A. Res. 1980, Vol. 8), polyacrylamide acryloylmorpholide, polydimethyl acrylamide polymerized on kieselfuhr (Nucleic Acid Res. 9(7) 1691 (1980)). Other supports described are of inorganic nature, in particular based on silica functionalized with a hydrocarbon radical bearing an NH2 and/or COOH group (JACS, 105, 661 (1983), or the support based on silica functionalized with a 3-aminopropyltriethoxysilane group whose use in phosphite and phosphoramidite synthesis for the preparation of oligonucleotides described for the first time in European patent No. 0,035,719.
Typically, when employing prior art solid supports, the first step in synthesizing nucleic acids consists of attaching the first nucleoside of the desired sequence to the solid support, traditionally consisting of glass beads of controlled porosity (CPG) or, more generally, of a functionalized organic or inorganic polymer bound to an A, T, C, G or U nucleoside, depending on whether the sequence to be prepared contains A, T, C, G, or U as the first deoxyribo-or ribonucleoside. Thus, commercially available automated reactors are equipped so that one of these nucleosides has already been attached to the support. The appropriate reactor is thus selected depending on whether the sequence begins with A, T, C, G, or U. Elongation of this first nucleoside then takes place in the 3xe2x80x2 to 5xe2x80x2 or 5xe2x80x2 to 3xe2x80x2 direction, by means of coupling reagents. One synthetic cycle, that is to say the coupling between two nucleotides, includes at least three steps: (1) deprotection of the 5xe2x80x2 or 3xe2x80x2 OH function of a first nucleotide, e.g., detritylation, (2) activation of the said 5xe2x80x2 or 3xe2x80x2 OH function of this nucleotide and condensation with the 3xe2x80x2 or 5xe2x80x2 end respectively of a second nucleotide, and, finally, (3) oxidation of the phosphite group of the internucleotide bond obtained to phosphate.
The oligonucleotide is preferable synthesized in the 3xe2x80x2 to 5xe2x80x2 direction. In this case, the staring material is a 5xe2x80x2 OH-protected nucleoside that is attached to the support via the 3xe2x80x2 end of the deoxyribose or ribose ring. The nucleotides which are subsequently added are in the form of a 5xe2x80x2-protected derivative whose 3xe2x80x2 hydroxyl possesses a substituted phosphite or phosphate group.
Various synthetic methods are currently employed and the are distinguished by the type of substitution on the phosphate. The phosphoramidite method, described, for example, in EP 0,061,746 and U.S. Pat. No. 4,458,066, is the preferred technique because of the high coupling yields achieved. In this method, a phosphoramidite group is attached to the 3xe2x80x2 hydroxyl. Besides the importance of these groups for the solubility of the nucleosides in the organic solvent, the phosphoramidite group renders the phosphorus atom more susceptible to attack by a primary hydroxyl function, such as that in the 5xe2x80x2 position of the detritylated growing nucleosides or chains. The deprotected 5xe2x80x2 hydroxyl function becomes sufficiently nucleophilic to react with the phosphoramidite group of the second nucleotide.
The oligonucleotides obtained at the end of the synthetic cycles must be detached from the support and the protective functions must be removed. Cleavage of the support, deprotection of the bases and removal of the group bonded to the phosphorus are carried out simultaneously in aqueous ammonia solution. In the case of RNA, ethanol makes it possible to solubilize the 2xe2x80x2-O-silyl-oligoribonucleotides, and to minimize the desilylation as native RNA is not stable in basic conditions. The aqueous ammonia/ethanol solution containing the oligoribonucleotide which has passed into the liquid phase is then separated from the glass support and evaporated. Removal of the silyl groups takes place in the presence of tetrabutylammonium fluoride (TBAF) at room temperature for sixteen hours. The TBAF is then neutralized with TEAA (triethylammonium acetate). Other methods include, for example, to so-called phosphotriester, phosphodiester, H-phosphonate, and phosphite methods.
As is apparent, despite the advantages that have been achieved in DNA and RNA syntheses, the art is in need of a multifunctional solid support that could be used to synthesize any oligonucleotide regardless of the nature of the 3xe2x80x2-terminal base.
The present invention is based, in part, on the development of universal solid supports suitable for use in synthesizing of oligonucleotides. The solid supports are universal in that they may be used irrespective of the first RNA or DNA nucleotide to be synthesized, and irrespective of the type of monomer reagent used during the synthesis, that is, irrespective of the type of substitution on the phosphate group in the 3xe2x80x2 position or in the 5xe2x80x2 position depending on whether the synthesis is carried out in the 5xe2x80x2xe2x86x923xe2x80x2 or 3xe2x80x2xe2x86x925xe2x80x2 direction.
The novel solid supports of the present invention can be employed in automated solid phase syntheses suing standard process conditions. In particular, with the present invention, the monomer reagent serving to attach the first nucleotide to the solid support should be a monomer reagent identical to the monomer reagent serving to attach the other nucleotides of the sequence during the synthesis, in particular as regards the 5xe2x80x2 protection and the 3xe2x80x2 protection.
With the present invention, the first nucleotide that is introduced contains a 3xe2x80x2 or 5xe2x80x2 phosphate group which is, after cleavage between the support and the oligonucleotide, under the usual conditions of deprotection in basic medium, capable at the end of the synthesis of liberating an end 3xe2x80x2 or 5xe2x80x2 OH. In one aspect, the invention is directed to multifunctional substrates suitable as a reagent for synthesizing oligonucleotide acids have the following formulae: 
One feature of the invention is that following synthesis of the oligonucleotide, deprotection of the protecting groups and cleavage of the oligonucleotide from the solid support is accomplished with treatment with a standard basic medium such as NH4OH, NaOH.